Sunday, March 31, 2013

Creating a pro-chondrogenic microenvironment

Ctrl-F for (*NEW*) for the new information. Several times cellulose is used in biomaterial scaffolds to induce chondrogenesis. Cellulose is also known as fiber and is not digested. The question is can we ingest enough of it to build up enough fiber in the bone marrow to build a pro-chondrogenic microenvironment? Since fiber is something that is studied in the mainstream and not just in the bubble of height increase I open the subject to all of you.How much fiber can build up in the bone marrow?

How do we create a pro-chondrogenic microenvironment within the epiphyseal bone marrow to allow for the formation of new growth plates? The goal of LSJL is to induce that environment.

"[We] assess the feasibility of the sequential application of extracellular matrix (ECM) and low oxygen to enhance chondrogenesis in human fetal synovium-derived stem cells (hfSDSCs). Human fetal synovial fibroblasts (hfSFs) include hfSDSCs, as evidenced by their multi-differentiation capacity and the surface phenotype markers typical of mesenchymal stem cells. Passage-7 hfSFs were plated on either conventional plastic flasks (P) or ECM deposited by hfSFs (E) for one passage. Passage-8 hfSFs were then reseeded for an additional passage on either P or E. The pellets from expanded hfSFs were incubated in a serum-free chondrogenic medium supplemented with 10 ng/ml transforming growth factor-β3 under either normoxia (21% O(2)) or hypoxia (5% O(2)) for 14 days. Pellets were collected for evaluation of the treatments (EE21, EE5, EP21, EP5, PE21, PE5, PP21, and PP5) on expanded hfSF chondrogenesis. Compared with seeding on conventional plastic flasks, hfSFs expanded on ECM exhibit a lower expression of senescence-associated β-galactosidase and an enhanced level of stage-specific embryonic antigen-4{The Extecellular Matrix has potential to reduce cellular senescence}. ECM-expanded hfSFs show increased cell numbers and an enhanced chondrogenic potential. Low oxygen (5% O(2)) during pellet culture enhances hfSF chondrogenesis. The presence of stem cells in hfSFs, and modulation of the in vitro microenvironment can enhance hfSDSC chondrogenesis."

The two elements of the microenvironment identified as being pro-chondrogenic are hypoxia and ECM. LSJL heavily upregulates ECM molecules.

Group PE5 had the most positive chondrogenic markers. Which would be groups first plated on conventional plastic flasks than transferred to ECM by synovial fibroblasts in an hypoxic environment. EE5 which is ECM for the whole time also had positive measures on all chondrogenic factors.

The reason that Plastic followed by ECM was better than pure ECM could be due to a catch-up growth phenomenon so ECM may be still better at all stages for chondrogenesis overall.

Cellular Response to Hypoxia("Any process that results in a change in state or activity of a cell (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of a stimulus indicating lowered oxygen tension. Hypoxia, defined as a decline in O2 levels below normoxic levels of 20.8 - 20.95%, results in metabolic adaptation at both the cellular and organismal level. ") genes for mus musculus also altered in LSJL:
Adam8{down}
Gnb1{down}

"Chondrocytes generate an integrated response to ECM stiffness and transforming growth factor β (TGFβ), a potent agonist of chondrocyte differentiation. Primary murine chondrocytes and ATDC5 cells{ATDC5 cells are progenitor cells to chondrocytes} grown on 0.5-MPa substrates deposit more proteoglycan and express more Sox9, Col2α1, and aggrecan mRNA relative to cells exposed to substrates of any other stiffness{0.5MPa is the optimal stiffness out of those tested to encourage chondrogenesis}. The chondroinductive effect of this discrete stiffness, which falls within the range reported for articular cartilage, requires the stiffness-sensitive induction of TGFβ1. Smad3 phosphorylation, nuclear localization, and transcriptional activity are specifically increased in cells grown on 0.5-MPa substrates{The benefits of this ECM stiffness may be due to an increase in Smad3 phosphorylation}. ECM stiffness primes cells for a synergistic response, such that the combination of ECM stiffness and exogenous TGFβ induces chondrocyte gene expression more robustly than either cue alone through a p38 mitogen-activated protein kinase-dependent mechanism."

"Upon integrin binding to ECM ligands and the generation of internal cell tension, cells develop focal adhesions, a highly ordered array of proteins including focal adhesion kinase (FAK), talin, vinculin, and α-actinin. These proteins interact with small GTPases (i.e., Rho, Rac) and other signaling pathways, facilitating changes in cytoskeletal organization, actinomyosin contractility, and cell shape with even small changes in matrix compliance"

"The activated TβRI phosphorylates Smad2 and Smad3 on the C-terminal domain, causing heteromerization with Smad4 and preferential retention in the nucleus, where Smads act as transcription factors. In chondrocytes, phosphorylated Smad3 recruits CBP to activate Sox9-mediated transcription of Col2α1"

"there is an optimal level of ROCK activity on 0.5-MPa substrates that activates chondroinduction, in part, through the induction of TGFβ1 expression on compliant substrates."

"BMP-inducible nuclear translocation of Smad1/5/8 requires sufficient ROCK-dependent cytoskeletal tension. ROCK can also enhance the activity of both Smad3 and Sox9 by phosphorylation of the Smad3 linker region or Sox9 on serine 181"

"MSCs [may] exist in a perivascular[near or around blood vessels] location and share a number of cell surface markers with pericytes"

"MSC show a greater propensity to differentiate to cartilage and bone, if they are bone marrow derived and to differentiate to fat, if isolated from adipose tissue"

"In order to self renew, stem cells need to be protected from differentiation signals and from apoptosis, and the niche provides the adhesion molecules, soluble factors and conditions that allow this in a concerted fashion. These soluble factors and ECM components activate various kinase cascades including the ERK1/2 MAPK and the PI-3K pathways"<-So not only do we need to induce differentiation signals to induce stem cells to chondrogenesis we need to protect them from differentiation signals that induce differentiation to other cell types.

"EGF, which stimulates the EGFR1 on MSCs, strongly activates the MAP kinases ERK1/2 and Jnk1, the Stat3 and PKC pathways and weakly stimulates the PI-3K pathway, and high concentrations of EGF induce osteogenic differentiation in MSCs"<-Thus we want to avoid high levels of EGF.

"High concentrations of covalently tethered EGF, which restrict signaling to the cell surface, result in increased osteogenic differentiation of MSCs, while low concentrations of soluble EGF, which induce receptor internalization, are anti-osteogenic"<-Reducing EGF levels may be part of the way to grow taller.

"Concomitant PI-3K stimulation prevents ERK1/2-dependent osteogenic differentiation."<-LSJL likely stimulates both PI3K and ERK1/2 which may be why it is more pro-chondrogenic than osteogenic.

"When chondrogenesis was assessed in cross-linked methacrylated hyaluronic acid hydrogels, the macromer density influenced MSC chondrogenesis: high density macromers resulted in increased chondrogenesis, but of inferior quality than seen with lower density gels"

"early MSC progenitors, defined by their smaller size and expression of podocalyxin-like protein (PODXL), selectively express alpha4 and alpha6 integrins, which are lost during culture. Freshly isolated MSC do not express the vitronectin receptor alphav beta5, but express low levels of the fibronectin receptor alpha5beta1 and the collagen receptors alpha1beta1, alpha2beta1 and alpha3beta1. Upon culture alphavbeta5 is up-regulated. Increased expression of alphavbeta5 and alpha5beta1 is observed during chondrogenic differentiation"

"MSCs do not seem to circulate in the vasculature under physiological conditions, it seems likely that they are released into the vasculature, when there is increased demand for these cells during any kind of tissue injury "

"Although MSCs accumulate in damaged tissue to some degree, recruitment of circulating MSC is very inefficient"

"MSCs seem to be resistant in general to certain apoptotic pathways perhaps due to very low expression of caspase 8 and caspase 9"

"Bone marrow (BM)-derived stem and progenitor cell functions including self-renewal, differentiation, survival, migration, proliferation, and mobilization are regulated by unique cell-intrinsic and -extrinsic signals provided by their microenvironment. Reactive oxygen species (ROS), especially hydrogen peroxide (H(2)O(2)), play roles in regulating stem and progenitor cell functions in various physiologic and pathologic responses. The low level of H(2)O(2) in quiescent hematopoietic stem cells (HSCs) contributes to maintaining their "stemness," whereas a higher level of H(2)O(2) within HSCs or their niche promotes differentiation, proliferation, migration, and survival of HSCs or stem/progenitor cells.Major sources of ROS are NADPH oxidase and mitochondria. In response to ischemic injury, ROS derived from NADPH oxidase are increased in the BM microenvironment{mechanical loading stimulates ROS release from mitochondria creating a more hypoxic microenvironment}, which is required for hypoxia and hypoxia-inducible factor-1α expression and expansion throughout the BM. This, in turn, promotes progenitor cell expansion and mobilization from BM, leading to reparative neovascularization and tissue repair. Excess amounts of ROS create an inflammatory and oxidative microenvironment, which induces cell damage and apoptosis of stem and progenitor cells."

"ROS such as O2•− and H2O2 are generated from a number of sources including mitochondria, NADPH oxidases (NOXs), xanthine oxidase, cytochrome P450, and nitric oxide synthase (through its uncoupling). Because O2•− is produced from oxygen, the oxygen concentration or hypoxic condition has a significant impact on the total amount of ROS. The O2•− reacts with nitric oxide (NO) to generate peroxynitrite (OONO−), thereby inhibiting endothelial function, whereas it can be quickly converted to H2O2 by superoxide dismutases (SODs) such as MnSOD (SOD2) or Cu/ZnSOD (SOD1) or extracellular SOD (SOD3). H2O2 is catalyzed by catalase, glutathione peroxidases (GPx′s), and the thioredoxin–peroxiredoxin system to nonreactive water"

"Growth factor signaling is mediated through H2O2 production."

"Different from phagocytic NADPH oxidase that is normally quiescent but generates a large burst of O2•− (the “oxidative burst”) upon activation, the NOXs constitutively produce low levels of O2•− or H2O2 intracellularly in the basal state and are further stimulated acutely by various agonists and growth factors. NOXs are now recognized to have specific subcellular localizations, which is required for localized H2O2 production and activation of specific redox signaling pathways to mediate various functions" NOXs are located in MSCs.

"Once O2•− is generated, it is immediately converted into H2O2 by MnSOD or Cu/ZnSOD"

"To avoid the potential damaging effects of H2O2, mitochondria express other antioxidant enzymes such as peroxiredoxin (Prx) 3 and Prx5 and glutathione peroxidase."

"optimal levels of ROS are required for normal responses, whereas excess or insufficient levels of ROS are associated with cellular dysfunction and reduced growth factor signaling, respectively"<-So we want to maximize ROS signaling before cellular dysfunction.

"PTEN is a negative regulator of the PI3K–Akt pathway and contains catalytic cysteine residues that are highly susceptible to oxidation by H2O2. Therefore, PTEN inhibition stabilizes the active phosphorylated form of Akt. "

(*NEW*)Directing chondrogenesis of stem cells with specific blends of cellulose and silk.

"We systematically prepared cellulose, blends with silk at different compositions using a method based on ionic liquids as a common solvent. We tested the effect of blend compositions on the physical properties of the materials as well as on their ability to support mesenchymal stem cell (MSC) growth and chondrogenic differentiation. The stiffness and tensile strength of cellulose was significantly reduced by blending with silk. The characterised materials were tested using MSCs derived from four different patients. Growing MSCs on a specific blend combination of cellulose and silk in a 75:25 ratio significantly upregulated the chondrogenic marker genes SOX9, aggrecan and type II collagen in the absence of specific growth factors{so the stiffness of this environment is likely optimal for chondrogenesis}. This chondrogenic effect was not found with neat cellulose or cellulose/silk 50:50 blend composition. No adipogenic or osteogenic differentiation is detected on the blends suggesting that cellulose/silk 75:25 blend induces specific stem cell differentiation into the chondrogenic lineage without addition of the soluble growth factor TGF-β."

"Cellulose is a linear homopolymer of glucose." Cellulose is also called fiber. It can't be digested so if you can get it to your bone marrow it may help create a pro-chondrogenic microenvironment.

"Cellulose, which comprises three hydroxyl groups per repeating unit, is theoretically a good choice as an initiator of chondrogenesis"

The study Galactooligosaccharides improve mineral absorption and bone properties in growing rats through gut fermentation. found that "Galactooligosaccharides (GOS), prebiotic nondigestible oligosaccharides derived from lactose" had no effect on femur length.

The emergence of mechanoregulated endochondral ossification in evolution.

"stable fractures with small gaps between bone ends can undergo intramembranous healing, involving ossification on a fibrous membrane; in larger fracture gaps and more straining mechanical environments, endochondral ossification is necessary for healing where a cartilage matrix is laid down which later, if the soft tissue succeeds in stabilising the fracture, provides a template for ossification. It is likely that endochondral bone emerged in evolution because it confers a greater fitness in a more demanding mechanical environment."So we want shear strain and fluid velocity at an amount that causes cartilage tissue to form.